Method for increasing neutrophil populations using in vitro-derived human neutrophil precursor cells

ABSTRACT

A suspension comprising human neutrophil precursor cells, wherein the cellular component is comprised of at least about 16% human myeloblasts and promyeclocytes, which have been derived from neutrophis progenitor cells obtained from peripheral blood, bone marrow or cord blood, and less than about 5% colony forming units (CFU) of at least about 50 cells is provided. An alternative suspension comprising human neutrophil precursor cells, wherein the cellular component is comprised of at least about 16% CD15+CD11b- cells and less than about 5% colony forming units (CFU) of at least about 50 cells also is provided, wherein at least about 60% of the CD15+CD11b- cells are myeloblasts and promyelocytes. The suspensions of the invention are useful in methods for increasing neutrophil populations in a patient having a reduced populations of neutrophils.

This application is a divisional of application Ser. No. 08/324,361,filed Oct. 14, 1994 now abandoned which is a continuation of applicationSer. No. 7/885,295 filed Mar. 23, 1992 now abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an in vitro-derived population of humanneutrophil precursor cells and to the use of this population of cells inclinical and research applications.

BACKGROUND OF THE INVENTION

The main infection and disease-fighting cell of the human immune systemis the white blood cell (leukocyte), which circulates through the blood.Approximately 50 to 65 percent of all leukocytes are a type of cellcalled a "neutrophil," which mediates much of the infection-fightingcapability of the white cells. When a human has a substantially lowerthan normal number of circulating 20 neutrophils, the patient isconsidered to be suffering from "neutropenia," i.e., a conditioncharacterized by an abnormally low number of circulating neutrophils.

A patient suffering from neutropenia then is at substantial risk frominfection and disease, as the diminished number of neutrophilscirculating in the blood substantially impairs the ability of thepatient to fight any infection or disease, as less neutrophils areavailable to engage in the battle. In severe cases of neutropenia theremay be essentially no neutrophils available to fight infection anddisease.

Neutropenia, itself, may be the result of disease, genetic disorders,drugs, toxins, and radiation as well as many therapeutic treatments,such as high dose chemotherapy (HDC) and conventional oncology therapy.For example, many cancers have been found to be sensitive to extremelyhigh doses of radiation or anti-neoplastic (anti-cancer) drugs. Thesecancers include malignant melanoma, carcinomas of the stomach, ovary,and breast, small cell carcinoma of the lung, and malignant tumors ofchildhood (including retinoblastoma and testicular carcinoma), as wellas certain brain tumors, particularly glioblastoma. However, suchintensive HDC is not widely used because it frequently causes such acompromise of the hematopoietic system that the result is death due toany of numerous opportunistic infections.

The reason behind the compromise, if not devastation, of thehematopoietic system resulting from HDC is generally understood. The HDCacts upon rapidly proliferating cells in the bone marrow that produceneutrophils, platelets, erythrocytes, lymphocytes, and other leukocytes.When the hematopoietic system is functioning correctly, platelets andneutrophils proliferate rapidly and turn over at a high rate, unlike thelymphocytes and red blood cells, which are long-lived. The result ofHDC, then, is that not only are cancerous (neoplastic) cells destroyed,so are the cells of the hematopoietic system that are responsible forgenerating the army of neutrophils that are necessary to maintain afunctioning immune system. Complete destruction of neutrophil progenitorand precursor cells eliminates the patient's short-term capacity togenerate mature neutrophils, thereby severely compromising the patient'sability to combat infection. The patient then becomes"immunocompromised" and subject to opportunistic infection. Such acondition may ultimately result in morbidity and death. Other situationsalso may be encountered where there has been a severe insult to thehematopoietic system, resulting in a substantial reduction inneutrophils and precursors thereto.

In order to understand the problems presented by neutropenia, whethercaused by HDC or otherwise, it is first necessary to understand somebasic principles about human blood cells, including their source andtheir development.

Hematopoiesis refers to the proliferation and differentiation of bloodcells. The major site of hematopoiesis in humans, after about 20 weeksof fetal life, is the bone marrow. Blood cells develop from multipotentstem cells that are usually located in the bone marrow. These stem cellshave the capacity to proliferate and differentiate. Proliferationmaintains the stem cell population, whereas differentiation results inthe formation of various types of mature blood cells that are groupedinto one of three major blood cell lineages, the lymphoid, myeloid orerythroid cell lineages. The lymphoid lineage is comprised of B cellsand T cells, which collectively function in antibody production andantigen detection, thereby functioning as a cellular and humoral immunesystem. The myeloid lineage, which is comprised of monocytes(macrophages), granulocytes (including neutrophils), and megakaryocytes,monitors the bloodstream for antigens, scavenges antigens from thebloodstream, fights off infectious agents, and produces platelets, whichare involved in blood clotting. The erythroid lineage is comprised ofred blood cells, which carry oxygen throughout the body.

The stem cell population constitutes-only a small percentage of thetotal cell population in the bone, marrow. The stem cells as well ascommitted progenitor cells destined to become neutrophils, erythrocytes,platelets, etc., may be distinguished from most other cells by thepresence of the particular progenitor "marker" antigen that is presenton the surface of these stem/progenitor cells. A group of antibodiesthat is capable of recognizing this particular marker antigen isreferred to as "cluster of differentiation 34" or "CD34". Thedesignation "CD34+" is used to describe a cell as one that has theparticular cell surface antigen that is recognized by the CD34 group ofantibodies. Stem cells, then, are CD34+. The majority of cells that areCD34+in bone marrow, however, are B lymphocyte progenitor cells andmyeloid progenitor cells. Neutrophils differentiate from stem cellsthrough a series of intermediate precursor cells, which can bedistinguished by their microscopic morphological appearance, includingsuch characteristics as the size of their nuclei, the shape of theirnuclei, cell size, nuclear/cytoplasmic ratio, presence/absence ofgranules, and staining characteristics (see FIG. 1 and Atlas of BloodCells: Function and Pathology, second edition, Zucker-Franklin et al.).Initially, the multipotent stem cell, which cannot be measured directlyin vitro, gives rise to myeloid "progenitor cells" that generateprecursors for all myeloid cell lines. The first myeloid progenitor isdesignated CFU-GEMM for "colony forming unit,--granulocyte, erythroid,macrophage and megakaryocyte". The CFU-GEMM progenitor, in turn, willgive rise to a CFU-GM progenitor cell, which is otherwise known as"colony forming unit--granulocyte and macrophage". In all of thesedescriptive terms, "colony" refers to a cell that is capable of givingrise to more than 50 cells as measured in 14 day in vitro assays forclonal growth, under conditions as set forth in Example 5 of the presentspecification. These cells will divide at least six times.

The CFU-GM is a committed progenitor--in other words, it is committed todifferentiating into granulocytes and macrophages only. It is neithercapable of differentiating into other types of cells nor is it capableof dedifferentiating into earlier stage progenitor cells. The CFU-GMprogenitor cell may then differentiate into a myeloblast. The timerequired for differentiation from a CFU-GEMM to a myeloblast is believedto be about 1-4 days. A myeloblast is the first of the series of cellsthat may be referred to as "precursors" to the neutrophils, as suchcells, once allowed to fully develop (differentiate), can only formneutrophils, which are only capable of undergoing fewer than six celldivisions and, therefore, do not form colonies in in vitro colony assaysas described previously.

Once differentiation has progressed to the myeloblast stage, themyeloblasts undergo terminal differentiation into promyelocytes, which,in turn, differentiate into myelocytes over a course of about 4-6 days.Within another 5 days or so, myelocytes differentiate intometamyelocytes, which, in turn, differentiate into banded neutrophils.These banded neutrophils finally differentiate into mature, segmentedneutrophils, which have a half-life of about 0.3 to 2 days. The term"progenitor" will be used to refer to stem cells, and cells which canform colonies. "Precursor" will be used to refer to myeloblasts,promyelocytes and myelocytes and, in some instances, metamyelocytes andbanded neutrophils, also.

During this progressive, morphologic differentiation, changes in thesurface antigens of these cells can be observed. For example, stemcells, CFU-GEMM and CFU-GM are CD34+. Hematopoietic cells thatdifferentiate beyond the CFU-GM stage are no longer CD34+. Similarprogressions of expression are observed for the cell-surface antigensCD33 and CD45RA. All neutrophil precursor cells subsequent to thepromyelocyte precursor cells may be characterized as CD34-, CD33+,CD38+, CD13+, CD45RA-, and CD15+. More mature cells also may becharacterized as CD11+ and CD16+ Terstappen et.al. Leukemia 4;657, 1990.It should be appreciated, however, that such transitions in cell-surfaceantigen expression are gradual, rather than abrupt, wherein some cellsof a particular precursor cell type may be positive and other cells ofthe same type may be negative for a particular cell-surface antigen.Furthermore, the determination that a particular cell type is positiveor negative for a particular cell-surface antigen will depend, in part,upon the particular method used to make that determination. Thecharacterization of cell differentiation by cell-surface antigenexpression may be confirmed by other means of characterizing celldifferentiation, such as cell morphology.

Specific growth factors react with specific receptors on stem cells todirect their differentiation into committed progenitor cells. Thesefactors regulate the proliferation and differentiation of hematopoieticcells. At least four colony-stimulating factors (CSFs) are known tocooperate in the regulation of neutrophil production. These fourfactors, which are referred to as GM-CSF (granulocyte and macrophage),IL-3 (interleukin-3), G-CSF (granulocyte), and M-CSF (macrophage), whichis also known as CSF-1, are synthesized by macrophages, T cells,endothelial cells and other types of cells. The potential of aprogenitor cell to respond to a CSF is determined, in part, by thepresence of receptors on the surface of the cell for that particular CSFand, in part, by the concentration of the particular CSF. There also issome indication for indirect stimulation, whether via an accessory cellor by synergistic action with other obligatory growth factors, such asc-kit ligand, IL-6 (interleukin-6), IL-11 (interleukin-11), IL-4(interleukin-4), and IL-1 (interleukin-1).

In addition to changes in morphology and cell-surface antigenexpression, as neutrophil precursor cells differentiate, they lose theircapacity to proliferate. In general, the less mature neutrophilprecursor cells, namely the myeloblasts, promyelocytes, and myelocytes,retain their ability to proliferate. However, the more matureneutrophils, namely the metamyelocytes and the banded neutrophils, losetheir capacity to proliferate, although they continue to differentiateinto mature, segmented neutrophils.

Several methods of treatment have been proposed to combat HDC-inducedneutropenia. These methods can partially ameliorate the neutropenia butcannot eliminate it completely. Bone marrow cells alone have been usedto provide the cellular component necessary for neutrophil recovery.However, this particular method of treatment only reduces the period ofneutropenia to about 2-3 weeks.

Several problems are associated with the use of bone marrow toreconstitute a compromised hematopoietic system. First, the number ofstem cells in bone marrow is very limited. Stem and progenitor cellsmake up a very small percentage of the nucleated cells in the bonemarrow, spleen, and blood. About ten times fewer of these cells arepresent in the spleen relative to the bone marrow, with even lesspresent in the adult blood.

As an example, approximately one in one thousand nucleated bone marrowcells is a progenitor cell; stem cells occur at a lower frequency.Secondly, a significant period of time is necessary for a stem cell todifferentiate to a mature neutrophil, on the order of at least 10-15days.

Bone marrow gathered from a different (allogeneic) matched donor hasbeen used to provide the bone marrow for transplant. Unfortunately,Graft Versus Host Disease (GVHD) and graft rejection limits bone marrowtransplantation even in recipients with HLA-matched sibling donors.Approximately half of the allogeneic bone marrow transplantationrecipients develop GVHD. Current therapy for GVHD is imperfect and thedisease can be disfiguring and/or lethal. Thus, risk of GVHD restrictsthe use of bone marrow transplantation to patients with otherwise fataldiseases, such as malignancies, severe aplastic anemia, thalassemias,and congenital immunodeficiency states. About 7,000 of the 15,000 bonemarrow transplantations performed each year are allogeneic. Many otherpatients have diseases that might be treated by marrow celltransplantation (such as sickle cell anemia) if GVHD or graft rejectionwere not such serious risks.

An alternative to allogeneic bone marrow transplants is autologous bonemarrow transplants. In autologous bone marrow transplants, some of thepatient'own bone marrow is harvested prior to treatment, such as HDC,and is transplanted back into the patient afterwards. Such a methodeliminates the risk of GVHD. However, autologous bone marrow transplantsstill present many of the same problems presented by allogeneic bonemarrow transplants in terms of the limited number of stem cells presentin the bone marrow and the amount of time required for a stem cell todifferentiate to a mature neutrophil. In addition, autologous marrowalso may be contaminated with tumor cells.

One approach to overcome the problems with bone marrow transplants hasbeen the attempted isolation of stem cells from donated bone marrow, orother sources, and the use of such stem cells to regenerate the immunesystem, such as after HDC. The theory behind this approach in theallogeneic setting is that the stem cell is naive in nature (has notdeveloped significant host-specific characteristics) and, therefore,will not be recognized in the transplant recipient as a foreign body orantigen, thus hopefully improving acceptance. Furthermore, since theseisolated cells contain minimal numbers of T-cells, it may be possible toavoid adverse reactions, as in GVHD.

Problems are also associated with this approach. Since the number ofstem cells in bone marrow is very limited and at least about 10-15 daysis required for stem cells to differentiate into mature neutrophils,significant in vivo multiplication of the cells must take place in orderto generate an adequate number of neutrophils for introduction into thepatient. Thus, the transplantation of stem cells at best results in animmunocompromised patient continuing to be immunocompromised for asignificant period of time.

Hematopoietic growth factors, such as G-CSF or GM-CSF, have beenadministered alone or in combination with autologous or allogeneictransplants of stem cell populations subsequent to HDC. Althoughneutrophils increase in number as a result of the treatment, the periodof severe neutropenia is only reduced to about ten days. Since theproduction of neutrophils from stem cells normally takes about 10-15days, stimulation of progenitor cell production and differentiation byhematopoietic growth factors and the eventual reconstitution of matureleukocytes, including mature neutrophils, requires a significant periodof time.

Peripheral blood stem cells (PBSC), which have been mobilized withchemotherapy or growth factors, also have been used to treatneutropenia. It is believed that the mobilized PBSC represent a mixtureof progenitor cells and, perhaps, precursor cells that occur naturallyduring the recovery of myelosuppressed bone marrow. Again, such amixture of progenitor and, perhaps, precursor cells only reducesneutropenia to about nine days. Furthermore, the precursor cells inthese mixtures probably would not survive freezing, since cellscontaining granules do not freeze well using presently known methods,and, therefore, could not be stored for subsequent treatments.

Generally speaking, none of these methods is successful in reducing theperiod of severe neutropenia below about 8-10 days. Such a lengthyperiod of neutropenia still renders the patient susceptible toinfection, the treatment of which requires hospitalization at asignificant cost.

Transfusions of mature neutrophils also have been attempted as a meansof addressing neutropenia. Such transfusions can be very expensive andinvolve healthy donors in a procedure that is time consuming,uncomfortable, and risky (Clift et al., symposium on InfectiousComplications of Neoplastic Disease (Part II), Vol. 76: 631-636 (1984).A major concern in the use of mature neutrophil transfusions is that, iftransfused mature neutrophils are unable to function and circulatenormally in the recipient individual, toxic reactions may result withadverse consequences (Wright, The American Journal of Medicine, Vol. 76:637-644 (1984).

U.S. Pat. No. 4,714,680 describes a suspension of humanlympho-hematopoietic stem cells substantially free of mature lymphoidand myeloid cells but which may further comprise colony forming cells.Such a composition could be used in the treatment of neutropenia,however, given the fact that the production of neutrophils from stemcells requires about 10-15 days, such a composition would not reduce theperiod of neutropenia.

U.S. Pat. No. 5,004,681 relates to hematopoietic stem and progenitorcells of neonatal or fetal blood that are cryopreserved, and thetherapeutic uses of such stem and progenitor cells upon thawing. Inparticular, the invention relates to the therapeutic use of fetal orneonatal stem cells for hematopoietic (or immune) reconstitution.

U.S. Pat. No. 5,061,620 describes a method to obtain a cellularcomposition of human hematopoietic stem cells, with fewer than 5% oflineage-committed cells. Such a composition also could be used in thetreatment of neutropenia but, once again, such a composition would notreduce the period of neutropenia, since 10-15 days would be required forneutrophils to differentiate from stem cells and CFU-GEMM.

U.S. Pat. No. 5,087,570 relates to concentrated hematopoietic stem cellcompositions that are substantially free of differentiated or committedhematopoietic cells. The cells are obtained by subtraction of cellshaving certain particular markers and selection of cells having otherparticular markers. The resulting composition may be used to provide forindividual or groups of hematopoietic lineages to reconstitute stemcells of the host, and to identify an assay for a variety ofhematopoietic growth factors.

There remains a need for an effective means of treatment tosignificantly reduce, if not completely eliminate, the period ofneutropenia. Such a treatment would enable a patient, who has undergoneHDC or some other form of chemotherapy, such as that associated withconventional oncology therapy, to combat infection, thereby reducing, ifnot completely eliminating, the risks of morbidity and death. Similarbenefits would be realized for patients suffering from drug, toxin,radiation or disease-induced neutropenia or genetic/congenitalneutropenia. In addition, such a treatment also could be used to treat apatient who, although not suffering from severe neutropenia, has areduced level of neutrophils.

SUMMARY OF THE INVENTION

The present invention provides a composition of human neutrophilprecursor cells, wherein the cellular component is comprised of at leastabout 16% myeloblasts and promyelocytes and less than about 5% colonyforming units (CFU) that give rise to at least about 50 cells.Alternatively, the cellular component of the composition may becomprised of at least about 16% CD15+CD11b- cells, at least about 60%which are myeloblasts and promyelocytes. The myeloblasts andpromyelocytes have the capacity to proliferate and differentiate intosegmented neutrophils. The neutrophil precursors may be purified so asto be substantially free of erythroid lineage-committed cells, includingBFU-E. The compositions may additionally comprise myelocytes,metamyelocytes, banded neutrophils, and/or segmented neutrophils. Themyelocytes, metamyelocytes, banded neutrophils, and/or segmentedneutrophils may be derived from the neutrophil precursor cells in vitro.The neutrophil precursor cells, themselves, may be derived fromperipheral blood, bone marrow, or cord blood.

The present invention also provides a method of treating a patientsuffering from neutropenia, which may result from HDC, conventionaloncology therapy, drugs, diseases, genetic disorders, toxins, andradiation, as well as a method of treating a patient who, although notsuffering from severe neutropenia, has a reduced population ofneutrophils. The method comprises the administration of a composition asdescribed above. The method of administration may be intravenous and maybe supplemented by the administration of stem cells and otherlineage-uncommitted cells.

The present invention further provides a method of gene therapy, whichcomprises the stable introduction of a gene, such as a gene forresistance to a chemotherapeutic drug or an absent or aberrantneutrophil constituent, into neutrophil progenitor cells, the subsequentin vitro culture of the neutrophil progenitor cells to proliferate anddifferentiate into neutrophil precursor cells, and the administration ofa composition comprising the genetically altered neutrophil precursorcells to a patient, who will be exposed to the chemotherapeutic drug, byintravenous injection, for example, or for correction of a neutrophilanomaly.

Alternatively, the gene may be introduced into the neutrophil precursorcells for administration to the patient.

Additional inventive features and advantages of the present inventionwill become apparent from the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Neutrophil differentiation in terms of cell morphology,cell-surface antigen expression, and transit time.

FIG. 2: Kinetics of a 26-day culture in which the total number of cellsincreased over 40 fold.

FIG. 3: CD15 and CD11b differentiation during a 21-day culture of CD34+cells.

FIG. 4: CD15 and CD11b sorted cells for determination of morphology asshown in FIG. 5.

FIG. 5: Morphology of CD15 and CD11b sorted cells as shown in FIG. 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an enriched population of humanneutrophil precursor cells. The enriched population of human neutrophilprecursor cells is derived from the in vitro culture of human stemand/or neutrophil progenitor cells. The human stem and/or neutrophilprogenitor cells may be obtained from bone marrow or peripheral bloodstem cells (PBSC), although other hematopoietic cell sources, whetherfetal, such as umbilical cord blood or liver, neonatal or adult, may beused. The particular source of human neutrophil progenitor cells willdepend, in part, upon the particular use to which the resultingpopulation of neutrophil precursor cells will be applied.

Although any source of human neutrophil progenitor cells may be used inresearch applications, bone marrow, in particular autologous bone marrowas opposed to allogeneic bone marrow, and peripheral blood CD34+ cellsare preferred sources of human neutrophil progenitor cells for thetherapeutic treatment of a patient undergoing oncology support or of apatient experiencing neutropenia subsequent to HDC. Such cell sourcesare also preferred for the therapeutic treatment of a patient sufferingfrom neutropenia that has been induced by a disease, drug, toxin, orradiation or from genetic neutropenia. Furthermore, such cell sourcesare also useful to treat patients who do not have neutropenia per se buthave reduced populations of neutrophils.

Bone marrow cells may be obtained from a source of bone marrow, such asthe iliac crest, tibia, femur, sternum, or another bone cavity. Bonemarrow may be aspirated from the bone in accordance with techniques thatare well known to those who are skilled in the art. The marrow may beharvested from a donor, in the case of an allogeneic transplant, or fromthe patient, himself, in the case of an autologous transplant. Themarrow may be processed as desired, depending mainly upon the useintended for the recovered cells.

White blood cells, in particular mononuclear cells (MNC), may becollected from the peripheral blood by leukapheresis. The MNC are thenpassed through a device containing a monoclonal antibody, such as CD34or other stem/progenitor recognition systems, linked to a solid phase.The CD34 monoclonal antibody binds CD34+ cells, which include neutrophilprogenitor cells, and the remainder of the cells pass through the devicewithout being bound. Once a sufficient number of neutrophil progenitorcells has been isolated, the patient is disconnected from the device.The advantage of such a method is that it allows extremely rareperipheral blood stem cells and progenitor cells to be harvested from avery large volume of blood, sparing the donor the expense and pain ofharvesting bone marrow and the associated risks of anesthesia,analgesia, blood transfusion, and infection.

The neutrophil progenitor cells obtained from other sources may beinitially separated from other cells by a relatively crude separation.Large numbers of lineage-committed cells, such as those cells committedto differentiate along erythroid and lymphoid cell lineages, may beremoved, if desired. However, it will be appreciated by one who isskilled in the art that it is not necessary to remove any or everyundesired class of lineage-committed cells from the neutrophilprogenitor cells. Since some form of positive selection may be employedin any purification protocol, the undesired lineage-committed cellswould not be selected. It is preferred that there be some form ofnegative selection for all of the undesired lineage-committed cellsinitially so that the number of such cells in a final positive selectionof neutrophil progenitor cells is minimized. By using a combination ofnegative selection, i.e., cell removal, with positive selection, i.e.,cell isolation, a substantially homogeneous population of neutrophilprogenitor cells may be obtained.

Various techniques may be used to separate the neutrophil progenitorcells from other lineage-committed cells. For relatively crudeseparations, i.e., separations where up to 10%, usually not more thanabout 5%, generally not more than about 1%, of the total cells presenthave the positively selected marker, e.g., CD34, various techniques ofdiffering efficacy may be employed. The separation techniques employedshould maximize the retention of viability of the fraction of the cellsto be collected. The particular technique employed will, of course,depend upon the efficiency of separation, cytotoxicity of the method,the ease and speed of separation, and what equipment and/or technicalskill is required.

Separation procedures may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents, either joined to a monoclonal antibody or used in conjunctionwith complement, and "panning", which utilizes a monoclonal antibodyattached to a solid matrix, or another convenient technique. Antibodiesattached to magnetic beads and other solid matrices, such as agarosebeads, polystyrene beads, hollow fiber membranes and plastic petridishes, allow for direct separation. Cells that are bound by theantibody can be removed from the cell suspension by simply physicallyseparating the solid support from the cell suspension. The exactconditions and duration of incubation of the cells with the solidphase-linked antibodies will depend upon several factors specific to thesystem employed. The selection of appropriate conditions, however, iswell within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the CD34+ cells tobind to the solid-phase linked antibodies. The bound cells are thenseparated from the solid phase by any appropriate method, dependingmainly upon the nature of the solid phase and the antibody employed.

Antibodies may be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used with a fluorescence activated cell sorter (FACS), to enable cellseparation. A FACS employs a plurality of color channels, low angle andobtuse light-scattering detection channels, and impedance channels,among other more sophisticated levels of detection, to separate or sortcells. Any technique may be employed as long as it is not detrimental tothe viability of the desired cells.

The progenitor cells initially may be separated from other cells by thecell-surface expression of CD34. For example, CD34+ cells may bepositively selected by magnetic bead separation, wherein magnetic beadsare coated with CD34-reactive monoclonal antibody. The CD34+ cells thenmay be removed from the magnetic beads. Release of the CD34+ cells fromthe magnetic beads may be effected by culture release or other methods.Purity of the isolated CD34+ cells may be checked with a FACSCAN® flowcytometer (Becton Dickinson, San Jose, Calif.), for example, if sodesired. Enrichment of CD34+ cells is preferred to minimize the volumeof culture medium used and to remove accessory cells, both alive anddead or dying, which may produce factors that affect the subsequentproliferation and differentiation of the selected cells in culture.However, the enriched CD34+population of cells does not necessarily haveto be pure.

The resulting enriched population of neutrophil progenitor cells thenmay be cultured to proliferate and differentiate into neutrophilprecursor cells. Enriched preparations of CD34+ cells may be placed in asuitable medium in culture plates. Conveniently, the medium in cultureplates is one that is well-defined and enriched. An example of asuitable medium is McCoy's 5A culture medium (Sigma, St. Louis, Mo.),which additionally contains fetal bovine serum (Hyclone, Logan, UT),horse serum (Hyclone), hydrocortisone (Sigma), μ-thioglycerol, andgentamicin (Gibco). The culture medium should further comprisehematopoietic growth factors, such as recombinant IL-3 (rIL-3),recombinant G-CSF (rG-CSF), recombinant GM-CSF (rGM-CSF)(Amgen, ThousandOaks, Calif.), C-kit ligand (steel factor), and stem cell factor(Genzyme, Boston, Ma.). It will be appreciated by one who is skilled inthe art that other suitable culture media may be used as well as othersuitable hematopoietic growth factors in various combinations.

It is preferred that the culture medium not be replaced during theperiod of culturing, although the cultures should be fed at weeklyintervals. However, in some cases, it may be desirable to change theculture medium from time to time, at least about once or twice per week.

CD34+ enriched cell populations obtained from bone marrow, however, maycontain significant numbers of CD19+CD34+ cells that do not proliferateunder certain culture conditions, such as those described above. It willbe appreciated by one who is skilled in the art that a greaterproliferation of neutrophil precursor cells possibly could be obtainedusing bone marrow CD34+ cells that have been significantly, if notcompletely, depleted of CD19+ cells or, alternatively, CD34+ cells couldbe obtained from cord or peripheral blood, where the population of CD19+cells is greatly reduced.

At selected days during the culture period, cell 20 aliquots may beremoved and labeled with fluorescent-conjugated CD15 and CD11bmonoclonal antibodies for sorting in a flow cytometer, such as theFACSCAN® flow cytometer (Becton Dickinson, San Jose, Calif.), based onexpression of CD15 and CD11b cell-surface antigens. Cells sortedaccording to CD15 and Cd11b antigen expression may be additionallycharacterized according to morphology and potential for colony-formingunits. Cells may be characterized by morphology as myeloblasts,promyelocytes, myelocytes, metamyelocytes, banded neutrophils, segmentedneutrophils, promonocytes and monocytes. Colony assays may be conductedin methyl cellulose containing other media components and growth factorsto determine the existence of CFU-GM, CFU-M, BFU-E, and CFU-GEMM.

After culturing for an appropriate length of time, CD15+Cd11b- cells,which are at least about 60% myeloblasts and promyelocytes, may beisolated and utilized in the therapeutic treatment of patients sufferingfrom neutropenia. The neutrophil precursor cells may be administered inthe form of a composition, wherein the cellular component is comprisedof at least about 16% myeloblasts and promyelocytes and less than about5% colony forming units (CFU) of at least about 50 cells. Alternatively,the cellular component of the composition may be comprised of at leastabout 16% CD15+CD11b- cells, at least 60% of which are myeloblasts andpromyelocytes, and less than about 5% colony forming units (CFU) of atleast about 50 cells. The myeloblasts and promyelocytes have thecapacity to proliferate and differentiate into segmented neutrophils.The compositions may be purified to be substantially free of erythroidlineage-committed cells, including BFU-E. The compositions mayadditionally comprise myelocytes, metamyelocytes, banded neutrophils,and/or segmented neutrophils. The myelocytes, metamylocytes, bandedneutrophils, and/or segmented neutrophils may be derived from theneutrophil precursor cells in vitro.

The cellular component of the present inventive compositions is enrichedfor neutrophil precursor cells, in particular myeloblasts andpromyelocytes, over that which is found normally in bone marrow. Forexample, in adults, the upper limit of the combined ranges ofmyeloblasts and promyelocytes in number fraction as percent is 12.5%. Itis also 12.5% in newborns approaching infancy and preschool children. Itis 15.0% in infants and school-age children and only 10.0% in day-oldnewborns. (Geigy Scientific Tables, Vol 3, C. Lentner, ed. Ciba-Geigy,Basel, Switzerland. 1984.)

The composition may be administered intravenously to a patient requiringa bone marrow transplant in an amount sufficient to reconstitute thepatient's hematopoietic and immune systems. The composition may besupplemented with stem cells and other lineage-uncommitted cells.Precise, effective quantities can be readily determined by those who areskilled in the art and will depend, of course, upon the exact conditionbeing treated by the particular therapy being employed.

A survey of published reports indicates that the number of CFU-GMinfused for autologous bone marrow reconstitution in human patients canbe relied on as an indicator of the potential for successfulhematopoietic reconstitution (Spitzer, G., et al., 1980, Blood 55(2):317-323; Douay et al., 1986, Exp. Hematol. 14:358-365). By standardizingpublished data by patient weight, and assuming a patient weight of 150pounds (67.5 kilograms), the calculated number of CFU-GM needed forsuccessful hematopoietic reconstitution using autologous bone marrowcells ranges from 2-425×10⁴ /kg patient weight, with faster recoverynoted using greater than 10×10⁴ CFU-GM. Accordingly, it is anticipatedthat the administration of compositions of the present inventioncomprising an equivalent or greater number of neutrophil and/orneutrophil precursor cells, either alone or in combination withstem/progenitor cells, should result in the successful reconstitution ofa human hematopoietic system in even shorter time.

Because of the unique aspect of the present invention, in which theneutrophil precursor cells have the capacity to both proliferate anddifferentiate in culture and remain viable for an extended period oftime, it is possible to isolate an initial quantity of cells from the invitro culture and to administer that quantity of cells to the patient.After a period of time, one or more additional aliquots of viableneutrophil and/or neutrophil precursor cells may be isolated fromculture and administered to the patient.

The neutrophil precursor cells may also find use in the treatment ofneutropenia induced by a disease, drug, toxin or radiation, as well asgenetic or congenital neutropenia. For example, aberrant neutrophilprecursor cells may be treated by genetically modified autologous orallogeneic neutrophil precursor cells. Such gene therapy may involve theintroduction of a wild-type gene into the neutrophil precursor cell orits progenitor cell, either by homologous or random recombination, forexample. Similarly, drug resistance genes may be introduced intoneutrophil precursor cells or their progenitor cells to enableneutrophil precursor cells and subsequently differentiated, more matureneutrophil cells to be resistant to one or more drugs, in particular,the drugs used in chemotherapy. Such neutrophil cells could betransplanted into a patient, either before or while undergoingchemotherapy, thereby eliminating the risk of neutropenia induced bychemotherapeutic agents. Diseases other than those specifically relatedto neutrophils may be treated, where the disease is related to the lackof a particular secreted product, such as a hormone, enzyme, interferon,factor, etc. Production of the protein that parallels natural productionmay be attained, even though production of the protein will be in adifferent cell type from that which normally produces such a protein, byemploying the appropriate regulatory sequences for inducible geneexpression. Alternatively, a ribozyme, antisense or other message may beinserted into the neutrophils to inhibit particular gene products orsusceptibility to disease.

The neutrophil precursor cells also may be used to treat human patientswho are not severely neutropenic but who have reduced levels ofneutrophils. Such populations of cells could be used to supplementexisting reduced populations of neutrophils in the patient to the cellpopulation numbers . In general, a normal healthy range of neutrophilsis considered to be from about 1,800 neutrophils/μl blood to about 7,000neutrophils/μl blood. Some patients may have levels of neutrophils aslow as about 500 neutrophils/μl blood, yet clinically do not appear tobe ill. However, patients with neutrophil levels below about 500neutrophils/μl blood are considered to be neutropenic and at risk forinfections and fever.

The neutrophil precursor cells also may be used in the research of theproliferation and differentiation of neutrophils. For example, factorsassociated with proliferation and differentiation, such as hematopoieticgrowth factors, may be evaluated. In addition, cytokine combinations andextracellular conditions may be evaluated. Similarly, the cells,themselves, may be used to evaluate particular media and fluids for cellproliferative and/or differentiative activity; etc.

The neutrophil precursor cells possibly may be frozen in liquid nitrogenfor long periods of storage. The cells then may be thawed and used asneeded.

Cryoprotective agents, which can be used, include but are not limited todimethyl sulfoxide (DMSO) (Lovelock, J. E. and Bishop, M. W. H., 1959,Nature 183:1394-1395; Ashwood-Smith, M. J., 1961, Nature 190:1204-1205),hetastarch, glycerol, polyvinylpyrrolidine (Rinfret, A. P., 1960, Ann.N.Y. Acad. Sci. 85:576), polyethylene glycol (Sloviter, H. A. andRavdin, R. G., 1962, Nature 196:548), albumin, dextran, sucrose,ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe, A. W., etal., 1962, Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose,choline chloride (Bender, M. A., et al., 1960, J. Appl. Physiol.15:520), amino acids (Phan The Tran and Bender, M. A., 1960, Exp. CellRes. 20:651), methanol, acetamide, glycerol monoacetate (Lovelock, J.E., 1954, Biochem. J. 56:265), and inorganic salts (Phan The Tran andBender, M. A., 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tranand Bender, M. A., 1961, in Radiobiology Proceedings of the ThirdAustralian Conference on Radiobiology, Ilbery, P. L. T., ed.,Butterworth, London, p. 59 ).

Typically, the cells may be stored in 10% DMSO, 50% serum, and 40% RPMI1640 medium. Once thawed, the cells may be induced to proliferate andfurther differentiate by the introduction of the appropriatehematopoietic growth factors.

Alternatively, the neutrophil precursor cells could be allowed toimmediately proliferate and differentiate into mature neutrophil cellsin culture, by providing the appropriate growth factors. Matureneutrophils cultured in accordance with the present invention arecapable of being cultured for about 4-5 weeks. This is significantlylonger than the typical survival of freshly isolated neutrophils, whichis only 72 hours or less, even when cultured in the presence of growthfactors.

The following examples serve to further illustrate the present inventionbut are not intended to limit the scope of the invention.

EXAMPLE 1

This example describes a preferred method of enriching a population ofCD34+ cells by positive selection.

Mononuclear cells were isolated from bone marrow on 1.077 g/dlHistopaque (Sigma, St. Louis, Mo.). The cells were washed incalcium/magnesium-free phosphate-buffered saline (CMF-PBS, Gibco, GrandIsland, N.Y.) and were resuspended in Iscoves-modified Dulbecco's Medium(IMDM, Sigma) containing 2% fetal bovine serum (Hyclone, Logan, Ut.) toa concentration of 1'10⁷ cells/ml. The cells were then contacted withmagnetic beads, which were coated with sheep anti-mouse IgG antibodies(Dynal, Oslo, Norway), sensitized with 1 μg of the anti-CD34 monoclonalantibody QBEND 10 (Quantum Biosystems, England) and used to captureCD34+ cells from the cell suspension. Essentially, CD34+ cells werepositively selected as described by Strauss et al., Am. J. Ped. Hematol.Oncol. 13:217 (1991), with slight modification. The magnetic beads andcells were rotated for 30 minutes at 4° C. at 2.5 rpm at a bead:cellratio of 1:1 or 5:1.

Following CD34 selection, the bead-cell complexes were isolated using amagnetic tube holder (Fenwal Div., Irvine, Calif.). After a series ofwashes in CMF-PBS, the CD34+ cells were released from the beads byadding 50 U/ml of Chymodiactin® (Bootes Pharmaceutical Co.,Lincolnshire, Il.) in RPMI 1640 (Sigma) and incubating for 15 minutes ina water bath at 37° C.

The cells that were released from the beads were evaluated for CD34purity by staining with the monoclonal antibody to CD34, namelyFITC-8G12 (Fenwal), for 15 minutes on ice and quantitating the stainedcells with a FACSCAN® flow cytometer (Becton Dickinson, San Jose,Calif.) using a side scatter vs. fluorescence display. The CD34+ cellpopulation was resolved as a population having FITC fluorescence and lowside scatter.

The level of purity of CD34+ cells obtained in accordance with thisprocedure averaged 66±16% (mean ±1 S.D., n=7) with a range from about40% to about 93% CD34+ as shown in Table I.

                                      TABLE I    __________________________________________________________________________    PRODUCTION OF NEUTROPHIL PRECURSORS AND    MATURE NEUTROPHILS IN CULTURES OF CD34+ CELLS    Initial Cell         Fold %    %    Preparation  Day     increase                              CD15+                                   CD15+    Experi-        %   % CD19+                 Of      in   CD11b-                                   CD11b+    ment #        CD34+            of CD34+                 Culture Cell #                              Region B                                   Region C    __________________________________________________________________________    1   54  N.D. 12      8.3  2    79    2   69  N.D. 12      6.9  50   47    3   93  N.D. 12      3.3  11   74    4   55  49   11      2.7  52   30    5   79  58   12      12.2 41   39                 26      48   8    91    6   40  55   12      7.8  45   36                 26      46   8    91    7   72  73   10      3.7  58   12                 35      74   17   83    Mean        66  59   Mean (D10-12)                         6.4  37   45.3    S.D.        16  9    __________________________________________________________________________     N.D. = not determined

EXAMPLE 2

This example describes a preferred method of ring CD34+ cells in vitro.

The enriched preparations of CD34+ cells were placed into 4-well cellculture plates (Nunc, Thousand Oaks, Calif.) or 25-75 cm² flasks(Costar, Cambridge, Ma.) at an initial cell concentration of 1-2×10⁵cells/ml in McCoy's 5A culture medium (Sigma) containing 12.5% fetalbovine serum (Hyclone), 12.5% horse serum (Hyclone), 10 μMhydrocortisone (Sigma), 10 μM α-thioglycerol (Sigma) and 10 μ/mlgentamicin (Gibco). The recombinant growth factors rIL-3, rG-CSF, andrGM-CSF (Amgen, Thousand Oaks, Calif.) were added at concentrations of300 U/ml, 200 U/ml, and 300 U/ml, respectively.

The cultures were placed in a 5% CO₂ /5% O₂, 37° C., high humidityincubator and incubated up to 37 days under these conditions. Thecultures were fed at weekly intervals with the above-described culturemedium, without medium replacement, to return the cell concentration to1-2×10⁵ cells/mli

FIG. 2 shows that the CD34+ enriched cell population was capable ofextensive proliferation in vitro. Total cell numbers increased graduallyuntil day 10-12 at which time large increases were observed. Cellproliferation continued beyond 20 days in culture and cells generated inthese cultures were capable of being viably maintained for up to about37 days. The enriched CD34+ progenitor cells were capable ofproliferating an average of 6.4±3.4 fold in 10-12 days and 56±15.6 foldafter 26-35 days (see Table I). The proportion of CD34+ cells graduallydeclined such that, after 7-10 days of culture, less than about 5% ofthe cells were CD34+, indicating that the CD34+ neutrophil progenitorcells had differentiated into neutrophil precursor cells and mature,segmented neutrophils. The increase in mature, segmented neutrophils,evidenced by the increase in CD15+Cd11b+ cells, is shown in FIG. 3.

EXAMPLE 3

This example describes a method used to assess the changes in phenotypesof the cultured CD34+ cells.

On selected days during the culture of the CD34+ cells, aliquots of1-2×10⁵ cells were removed from the culture plate or flask and werewashed once or twice in phosphate-buffered saline containing 0.05%bovine serum albumin and 0.1% azide (PAB). The cells were then labeledwith CD15 (LeuM1) FITC-conjugated and CD11b (Leu15) PE-conjugatedmonoclonal antibodies (Becton Dickinson) for 10 minutes on ice. Afterone additional wash in PAB, the cells were suspended in 1 ml of PAB andanalyzed using the FACSCAN® flow cytometer (Becton Dickinson) forexpression of CD15 and CD11b.

Pre-enrichment bone marrow cells (day 0) contain a large population ofCD15+Cd11b+ cells, as shown in Figure 3 and region C of FIG. 4. Thispopulation of CD15+CD11b+ cells represents metamyelocytes, bandedneutrophils and segmented neutrophils. A smaller population ofCD15+CD11b- cells is also observed in bone marrow (FIG. 3 and FIG. 4,region B). This population of CD15+CD11b- cells represents promyelocytesand myelocytes.

Enriched CD34+ cell preparations (post-enrichment, day 0) retain some ofthe CD15+CD11b+ cells as shown in FIG. 3. However, after three days ofculture, a CD15+CD11b- population is observed as shown in FIG. 3. Thispopulation of CD15+CD11b- cells increases further by days 7-10 and fromdays 13-20 a CD15+CD11b+ population is observed. The CD15+CD11b+population is indicative of the maturation of the cultures to segmentedneutrophils. A CD15-CD11b+ population, which is shown in region D ofFIG. 4, was observed during days 3-13 of culture but, after the 13th dayof culture, this particular population of cells, which representsmonocytes or macrophages, was no longer present.

EXAMPLE 4

This example describes a method used to assess the changes in morphologyof the cultured CD34+ cells. The cells that were defined in Example 3,in terms of their expression of CD15 and CD11b, were sorted with aFACStar® flow cytometer and their morphology was identified.Approximately 10,000-30,000 cells were sorted into tubes or cytospinfunnels. Cytospin slides were prepared by centrifuging the cytospinfunnels at 600 rpm for 7 minutes, using a Shandon Cytospin 2(Pittsburgh, Pa.). The cells were then stained with a Wright-Giemsastain (Harleco, Gibbstown, N.J.) for 30 seconds. After staining, thecells were rinsed in a phosphate buffer (sigma) for one minute. Theslides were evaluated for the presence of myeloblasts, promyelocytes,myelocytes, metamyelocytes, banded neutrophils, and segmentedneutrophils, as well as for the presence of promonocytes and monocytes.

Morphological analysis of CD15 and CD11b sorted fresh marrow cells,shown in the left panel of FIG. 4, revealed blast cells and lymphocytesin region A (CD15-CD11b-), promyelocytes and myelocytes in region B(CD15+CD11b-), metamyelocytes, banded neutrophils, and segmentedneutrophils in region C (CD15+CD11b+), and monocytes in region D(CD15-CD11b+). These data are not shown.

Morphological analysis of Wright-Giemsa stained cytospin preparations ofCD15 and CD11b sorted CD34+ cells that had been cultured for 7 days,shown in the middle panel of FIG. 4, revealed blast cells, lymphocytesand early promyelocytes in region A (CD15-CD11b-), promyelocytes inregion B (CD15+CD11b-), metamyelocytes and banded neutrophils in regionC (CD15+CD11b+), and macrophages in region D (CD15-CD11b+). Analysis ofregion C in sorted cells that had been cultured for 35 days (FIG. 4,right panel) revealed the presence of mature neutrophils. These data areshown in FIG. 5, A-E respectively. These studies validated themorphological stages observed by flow cytometry and confirmed thesequential expression of CD15 and CD11b during the in vitrodifferentiation of neutrophils.

EXAMPLE 5

This example describes a colony assay, which is used to determine thetypes of proliferative cells that are present in the CD34+ culturedcells.

On selected days during the culture of CD34+ cells, aliquots of cellswere placed in 35 mm dishes (Nunc) containing methyl cellulose, Iscoves'IMDM (Sigma), 30% FBS (Sigma), 7% Leptalb 7 (Armour Pharmaceuticals,Kankakee, Il.) and the recombinant growth factors rIL-3, rGM-CSF,rG-CSF, rIL-6 and erythropoietin (Amgen) at concentrations of 150 U/ml,200 U/ml, 150 U/ml, 160 U/ml, and 10 U/ml, respectively, to a finalconcentration of 5-10×10³ cells/mi. Colony assays were set up intriplicate and the colonies (CFU or colony forming unit of about 50cells or more) were scored as either CFU-GM, CFU-M (colony formingunit--macrophage), BFU-E (burst forming unit--erythroid) or CFU-GEMM.

The number of CFU-GM colonies increased during the early part of thecultures to an average of 4.3 fold after 1 week or 5.6 fold after 2weeks. Peak increase in CFU-GM occurred around day 10, when the CFUpresent were predominantly CFU-GM. In contrast, the number of BFU-Egenerally declined to less than half of the original number by two weeksof culture. Similarly, the numbers of CFU-GEMM and CFU-M also declined.

EXAMPLE 6

This example describes a method used to assess the proliferativepotential and colony forming cells present in CD34+ cultured cells.

On selected days (9-14) during the culture of CD34+ cells aliquots ofthe cells were stained with antibodies that bind to CD15 and CD11b.Cells from the regions described in Example 3 were then sorted intocolony assays and into liquid cultures identical to those that had beenused before.

Shown in Table II are the results from four experiments. Cells fromregion A (CD11b-CD15-) continue to proliferate from about 2-5.7 foldafter 7 additional days of culture and contain from about 0.4-2% colonyforming cells. Cells from region B (CD11b-CD15+) continue to proliferatefrom about 9.5-12 fold and contain from about 1.1-3% non-erythroidcolony forming cells. Colony forming cells are not present in region Cand no proliferation takes place upon subsequent culturing. These dataindicate that the CD15+CD11b- cells contain less than about 5% colonyforming cells and are capable of proliferating about at least 10 fold.

                  TABLE II    ______________________________________    PROLIFERATION AND CFC PRESENT IN CD11b/CD15 PHENO-    TYPES FROM UMBILICAL CORD BLOOD ENRICHED CD34+    CELLS              Experiment Number              1       2        3        4    ______________________________________    % CD34      45        80       80     59    Day of Culture                11        14       9      9    Fold Change Cell #                *72       54       27     34    Region A (11b-                **5.7     2        ND     ND    15-) fold change    CFU-GM      ***19     14       8      29    CFU-M       38        14       25     97    BFU-E       17        14       39     60    CFU-MIX     0         0        6      22    Cloning Efficiency                0.55      0.42     0.78   2.08    Region B (11b-                9.5       12       ND     ND    15+) fold change    CFU-GM      41        64       11     27    CFU-M       69        188      295    246    BFU-E       0         0        0      0    CFU-MIX     0         0        0      0    Cloning Efficiency                1.1       2.52     3.06   2.73    Region C (11b+                1.1       ND       ND     ND    15+) fold change    CFU-GM      0         ND       9      0    CFU-M       0         ND       0      0    BFU-E       0         ND       0      0    CFU-MIX     0         ND       0      0    Cloning Efficiency                0         0        0.09   0    ______________________________________     ND = not determined     *Fold increase in cell number during initial culture period     **Fold increase in cell number from the sorted phenotype after an     additional 7 days of culture     ***Colonies per 10 4 cells of the sorted phenotype

The neutrophil precursor cells may be used in the therapeutic treatmentof neutropenia associated with HDC and other types of chemotherapy, suchas conventional oncology therapy, neutropenia induced by disease, drugs,toxins, radiation and other agents, genetic neutropenia, and in thetreatment of human patients who, although not suffering from severeneutropenia, have reduced populations of neutrophils. The neutrophilprecursor cells also may be used in the research of neutrophils, such asneutrophil proliferation and differentiation.

All publications and patent applications cited herein are herebyincorporated by reference to the same extent as if each individualdocument was individually and,specifically indicated to be incorporatedby reference.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. A method of treating a human patient having areduced population of neutrophils, which method comprises administeringto said patient an isolated suspension of human neutrophil precursorcells, wherein said suspension comprises at least about 16% CD15+CD11b-human neutrophil precursor cells and less than about 5% CD34+ colonyforming units, colony forming units wherein at least about 60% of saidprecursor cells are myeloblasts and promyelocytes.
 2. The method ofclaim 1, wherein said human patient is suffering from a neutropeniaselected from the group consisting of neutropenia associated with highdose chemotherapy (HDC), neutropenia associated with conventionaloncology therapy, drug-induced neutropenia, disease-induced neutropenia,genetic neutropenia, toxin-induced neutropenia, and radiation-inducedneutropenia.
 3. The method of claim 1, wherein said suspension isadministered intravenously.
 4. The method of claim 1, wherein saidCD15+CD11b- cells have the capacity to proliferate and differentiateinto segmented neutrophils.
 5. The method of claim 4, wherein saidcomposition is substantially free of erythroid lineage-committed cells,including Burst Forming Unit--Erythroid (BFU-E).
 6. The method of claim4, wherein said suspension additionally comprises one or more cell typesselected from the group consisting of myelocytes, metamyelocytes, bandedneutrophils, and segmented neutrophils.
 7. The method of claim 6,wherein said additional cell types are derived from said CD15+CD11b-cells in vitro.
 8. The method of claim 1, wherein said CD15+CD11b- cellsare derived from neutrophil progenitor cells obtained from a sourceselected from the group consisting of peripheral blood, bone marrow, andcord blood.
 9. The method of claim 1, which method additionallycomprises the co-administration of stem cells and other progenitorcells.
 10. The method of claim 1, wherein said suspension comprises atleast about 25% CD15+CD11b- human neutrophil precursor cells.
 11. Themethod of claim 1, wherein said suspension comprises at least about 37%CD15+CD11b- human neutrophil precursor cells.
 12. The method of claim 1,wherein said suspension is administered in a therapeutically effectiveamount to increase the rate of neutrophil reconstitution in saidpatient.